Protecting groups are critical and ubiquitous features of modern synthetic organic chemistry, due to the need to carry out site-specific transformations in the presence of potentially numerous similar reactive functionalities. Since the early 1980's, research into protective groups for virtually any reactive functionality—amine, alcohol, carbonyl, carboxylic acid, thiol, phosphate to name a few—has produced thousands of reagents having a wide variety of chemical stabilities and applications. Indeed, the choice of compatible and orthogonal protection for complex organic syntheses is often one of the key factors in designing a successful synthetic scheme (see, e.g., Greene's Protective Groups in Organic Synthesis, (Wuts et al., fourth edition, Wiley Interscience, John Wiley and Sons Inc., 2007) herein incorporated by reference).
The use of silyl protecting groups for the temporary blocking of reactive hydroxyl functionalities has become commonplace in synthetic organic chemistry. Groups such as trimethylsilyl, t-butyldimethylsilyl, phenyldimethylsilyl and triphenysilyl are routinely used for the protection of hydroxyl groups as silyl ethers in the preparation of simple alcohols as well as of complex natural products. These groups have the advantage of being removable by treatment with fluoride ion, a reagent to which most other protecting groups exhibit good to excellent stability.
Silyl ether protecting groups have also been applied to the synthesis of oligonucleotides (U.S. Pat. Nos. 5,889,136, 6,008,400, 6,111,086, 6,590,093; Scaringe, Methods 23, 206-217 (2001); Scaringe, et al., J. Am. Chem. Soc. 120, 11820-11821 (1998); herein incorporated by reference).
Despite the significant improvements realized with the 5′-silyl-2′-orthoester synthesis invention, 5′-silyl ether protecting groups described in the literature are not visibly colored and do not provide the convenient colorimetric capability, an attribute that is advantageous for assessing coupling efficiency and is a feature of, for example, the traditional dimethoxytrityl (or DMT) 5′-protecting group. Thus, currently available reagents do not allow for visual detection of the deprotection step (i.e, via release of the silyl protecting group) which would allow evaluation of the coupling step. Additionally, it is desirable that each deprotection solution be collected in its entirety and the quantity of the protecting group released determined spectrophotometrically. Each value so obtained could then be ratioed with the immediately preceding value to obtain a nearly quantitative measure of the coupling efficiency for each cycle.
Thus, a need exists for a complement to the efficient chemical synthesis of RNA utilizing the 5′-silyl-2′-orthoester synthesis platform with a colorimetrical assay to monitor the individual coupling efficiencies of each synthesis cycle.